David C. Wilton

Roles of Trp31 in High Membrane Binding and Proinflammatory Activity of Human Group V Phospholipase A2

Group V phospholipase A2 is a recently discovered secretory phospholipase A2(PLA2) that has been shown to be involved in eicosanoid formation in inflammatory cells, such as macrophages and mast cells. We have demonstrated that human group V PLA2(hsPLA2-V) can bind phosphatidylcholine (PC) membranes and hydrolyze PC substrates much more efficiently than human group IIa PLA2, which makes it better suited for acting on the outer plasma membrane (Han, S.-K., Yoon, E. T., and Cho, W. (1998)Biochem. J. 331, 353–357). In this study, we demonstrate that exogenous hsPLA2-V has much greater activity than does group IIa PLA2 to release fatty acids from various mammalian cells and to elicit leukotriene B4 formation from human neutrophils. To understand the molecular basis of these activities, we mutated two surface tryptophans of hsPLA2-V to alanine (W31A and W79A) and measured the effects of these mutations on the kinetic activity toward various substrates, on the binding affinity for vesicles and phospholipid-coated beads, on the penetration into phospholipid monolayers, and on the activity to release fatty acids and elicit eicosanoid formation from various mammalian cells. These studies show that the relatively high ability of hsPLA2-V to induce cellular eicosanoid formation derives from its high affinity for PC membranes and that Trp31 on its putative interfacial binding surface plays an important role in its binding to PC vesicles and to the outer plasma membrane.

A comparison of the molecular species compositions of mammalian lung surfactant phospholipids

While dipalmitoyl phosphatidylcholine (PC16:0/16:0) is essential for pulmonary surfactant function, roles for other individual molecular species of surfactant phospholipids have not been established. If any phospholipid species other than PC16:0/16:0 is important for surfactant function, then it may be conserved across animal species. Consequently, we have quantified, by electrospray ionisation mass spectrometry, molecular species compositions of phosphatidylcholine (PC), phosphatidylglycerol (PG) and phosphatidylinositol (PI) in surfactants from human, rabbit, rat and guinea pig lungs. While PC compositions displayed only relatively minor variations across the animal species studied, there were wide variations of PG and PI concentrations and compositions. Human surfactant PG and PI were enriched in the same three monounsaturated species (PG16:0/18:1, PG18:1/18:1 and PG18:0/18:1) with minimal amounts of PG16:0/16:0 or polyunsaturated species, while all animal surfactant PG contained increased concentrations of PG16:0/16:0 and PG16:0/18:2. Animal surfactant PIs were essentially monounsaturated except for a high content of PI18:0/20:4 (29%) in the rat. As these four surfactants all maintain appropriate lung function of the respective animal species, then all their varied compositions of acidic phospholipids must be adequate at promoting the processes of adsorption, film refinement, respreading and collapse characteristic of surfactant. We conclude that this effectively monounsaturated composition of anionic phospholipid molecular species is a common characteristic of mammalian surfactants.

The antibacterial properties of secreted phospholipases A2

There is a considerable body of evidence to support the antibacterial properties of the group IIa phospholipase A(2) as an important physiological function. This enzyme is able to act as an acute phase protein and may be part of the innate defence system of the body, acting in concert with other antibacterial proteins and peptides. The enzyme is most effective against Gram-positive bacteria whereas penetration of the lipopolysaccharide coat of Gram-negative bacteria requires bactericidal/permeability-increasing protein (BPI) as an additional permeabilizing factor. The global cationic nature of this protein (pI>10.5) appears to facilitate penetration of the anionic bacterial cell wall. In addition, the considerable preference of the enzyme for anionic phospholipid interfaces provides specificity toward anionic bacterial membranes as opposed to zwitterionic eucaryotic cell membranes.

Interfacial binding of secreted phospholipases A2: More than electrostatics and a major role for tryptophan

Secreted phospholipases A(2) have similar catalytic sites, but vastly different interfacial binding surfaces that modulate their binding affinity for different kinds of phospholipid vesicles by several orders of magnitude. The structure/function principles that dictate both the differential interfacial binding and the physiological function of these enzymes are beginning to be unraveled.

The organisation of cholesterol and ergosterol in lipid bilayers based on studies using non-perturbing fluorescent sterol probes.

The fluorescence properties of dehydroergosterol and cholesta-5,7,9-trien-3 beta-ol have been studied in organic solution, in aqueous dispersions and incorporated into aqueous lipid dispersions. The absorption spectra of aqueous dispersions of the probes are very different to those in organic solution, and aqueous dispersions are non-fluorescent. This can be attributed to micelle formation with dimerisation and/or aggregation in the micelles. Concentration quenching also occurs when sterols are incorporated into lipid bilayers, but relatively high fluorescence is observed even at a 1 : 1 steroid:lipid molar ratio. Further, the fluorescence is still polarized at these high molar ratios. We attribute this to the formation of ordered arrays of sterol molecules in the lipid bilayers. In these arrays the sterol molecules are organised in an end-to-end fashion, and face-to-face overlap of the sterols is prevented by the lipid molecules. Possible structures for 1 : 1 mixtures are presented.

Action of human group IIa secreted phospholipase A2 on cell membranes. Vesicle but not heparinoid binding determines rate of fatty acid release by exogenously added enzyme.

Human group IIa phospholipase A2 (hIIa-PLA2) is a highly basic protein that is secreted from a number of cells during inflammation and may play a role in arachidonate liberation and in destruction of invading bacteria. It has been proposed that rodent group IIa PLA2 is anchored to cell surfaces via attachment to heparan sulfate proteoglycan and that this interaction facilitates lipolysis. hIIa-PLA2 contains 13 lysines, 2 histidines, and 10 arginines that fall into 10 clusters. A panel of 26 hIIa-PLA2 mutants were prepared in which 1–4 basic residues in each cluster were changed to glutamate or aspartate (charge reversal). A detailed analysis of the affinities of these mutants for anionic vesicles and for heparin and heparan sulfate in vitro and of the specific activities of these proteins for hydrolysis of vesiclesin vitro and of living cell membranes reveal the following trends: 1) the affinity of hIIa-PLA2 for heparin and heparan sulfate is modulated not by a highly localized site of basic residues but by diffuse sites that partially overlap with the interfacial binding site. In contrast, only those residues on the interfacial binding site of hIIa-PLA2 are involved in binding to membranes; 2) the relative ability of these mutants to hydrolyze cellular phospholipids when enzymes were added exogenously to CHO-K1, NIH-3T3, and RAW 264.7 cells correlates with their relative in vitro affinity for vesicles and not with their affinity for heparin and heparan sulfate. 3) The rates of exogenous hIIa-PLA2-catalyzed fatty acid release from wild type CHO-K1 cells and two mutant lines, one lacking glycosaminoglycan and one lacking heparan sulfate, were similar. Thus basic residues that modulate interfacial binding are important for plasma membrane fatty acid release by exogenously added hIIa-PLA2. Binding of hIIa-PLA2 to cell surface heparan sulfate does not modulate plasma membrane phospholipid hydrolysis by exogenously added hIIa-PLA2.

Microsomal fatty acyl-CoA transacylation and hydrolysis: fatty acyl-CoA species dependent modulation by liver fatty acyl-CoA binding proteins.

arachidonoyl-CoA. In summary, the data established for the first time a role for both L-FABP and ACBP in microsomal phosphatidic acid biosynthesis. By preferentially stimulating microsomal transacylation of unsaturated long chain fatty acyl-CoAs while concomitantly exerting their differential protection from microsomal acyl-CoA hydrolase, L-FABP and ACBP can uniquely function in modulating the pattern of fatty acids esterified to phosphatidic acid, the de novo precursor of phospholipids and triacylglycerols. This may explain in part the simultaneous presence of these proteins in cell types involved in fatty acid absorption and lipoprotein secretion.

Bacterial cell membrane hydrolysis by secreted phospholipases A2: a major physiological role of human group IIa sPLA2 involving both bacterial cell wall penetration and interfacial catalysis

The ability of human group IIa secreted phospholipase A(2) (human sPLA(2)) to hydrolyse the phospholipid membrane of whole cell suspensions of Gram-positive bacteria is demonstrated in real time using a continuous fluorescence displacement assay. Micrococcus luteus is used as a model system and demonstrates an almost absolute specificity for this human enzyme compared with porcine pancreatic and Naja naja venom sPLA(2)s. This specificity is due to selective penetration of the highly cationic human sPLA(2)50%) phospholipid hydrolysis was observed and this was confirmed by electrospray mass spectrometry that allowed the identification of several molecular species of phosphatidylglycerol as the targets for hydrolysis. However, the bactericidal activity of the human enzyme under these assay conditions was low, highlighting the capacity of the organism to survive a major phospholipid insult. In addition to pure enzyme, the human sPLA(2) activity in tears was demonstrated using M. luteus as substrate. In comparison to M. luteus, cell suspensions of Staphylococcus aureus were highly resistant to hydrolysis by human sPLA(2) as well as to the pancreatic and venom enzymes. Treatment of this organism with the specific cell wall protease lysostaphin resulted in a dramatic enhancement in cell membrane phospholipid hydrolysis by all three sPLA(2)s. Overall, the results highlight the potential of the human sPLA(2) as a selective antimicrobial agent against Gram-positive bacteria in vivo because this enzyme is essentially inactive against mammalian plasma membranes. However, the enzyme will be most effective in combination with other antimicrobial agents that enhance the permeability of the bacterial cell wall and where potentiation of the effectiveness of other antibiotics would be expected.

Human non-pancreatic (group II) secreted phospholipase A2 expressed from a synthetic gene in Escherichia coli: characterisation of N-terminal mutants

A gene coding for human non-pancreatic (group II) secreted phospholipase A2 (hnpsPLA2) has been constructed by the single-step ligation of twelve synthetic oligonucleotides. The gene has been cloned into a modification of the bacterial expression vector pET 11 which allows protein over-expression as inclusion bodies and enables about 3 mg/litre of pure refolded fully active enzyme to be obtained. The protein was expressed as a 1-Ala mutant (N1A) to allow removal of the initiator methionine by the Escherichia coli amino-peptidase. This mutant had very similar properties to the wild-type enzyme. A double mutant, N1A, V3W was also constructed and expressed in high yield. This tryptophan-containing mutant showed similar properties to the wild-type and N1A mutant but had about 40% of the activity under the assay conditions used. This tryptophan was used as a reporter group for interfacial binding and its properties were compared to those of the corresponding tryptophan in PLA2 from procine pancreas. Expression of the wild-type gene sequence for hnpsPLA2 in E. coli gave the expected mutant protein still with the initiator methionine and with much reduced activity. Interfacial binding of all hnpsPLA2 mutants to anionic phospholipids was very similar when assessed by fluorescence methods. Comparisons of these mutants with the pancreatic enzyme revealed significant differences in terms of the effect of calcium on interfacial binding. The ability to express reasonably large amounts of the N1A mutant in E. coli will provide a basis for future site directed mutagenesis studies of this important human enzyme.

Catalytic and non-catalytic functions of human IIA phospholipase A2

Group IIA phospholipase A2 (PLA2) is a low-molecular-mass secreted PLA2 enzyme that has been identified as an acute phase protein with a role in the inflammatory response to infection and trauma. The protein is possibly unique in being highly cationic and having a global distribution of surface arginine and lysine residues. This structure supports two functions of the protein. (1) An anti-bacterial role where the enzyme is targeted to the anionic cell membrane of Gram-positive bacteria and phospholipid hydrolysis assists in bacterial killing. (2) A proposed non-catalytic role in which the protein forms supramolecular aggregates with anionic phospholipid vesicles or debris. These aggregates are then internalized via interactions with cell surface heparin sulphate proteoglycans and macropinocytosis for disposal by macrophages.